Caco-2 cell cultures in the assessment of intestinal absorption: Effects of some co-administered drugs and natural compounds in biological matrices

Leena Laitinen

Doctoral dissertation, June 2006.
University of Helsinki, Faculty of Pharmacy, Division of Biopharmaceutics and Pharmacokinetics.

Several different in vitro absorption models are used in the screening of new drug candidates. One of them is the Caco-2 model, a widely used in vitro model for small intestinal absorption. Caco-2 cells, which originate from a colon carcinoma, differentiate spontaneously to cells that resemble mature small intestinal enterocytes and express carrier proteins similar to the small intestine, and can therefore be used for the assessment of active transport processes during intestinal absorption. Due to variation in cell line differentiation and selection of sub populations, permeability data obtained from different laboratories is seldom directly comparable. Hence, the use of several drugs or compounds with known permeability characteristics are recommended for model standardisation and the use of internal standards in every experiment could offer a solution to the problem.

In this study, the simultaneous use of five to ten drugs as internal standards was evaluated. Drugs with different permeability characteristics (high and low permeability, passive and active transport and active efflux) were used to detect their possible effects on cell viability, monolayer integrity and possible interactions during permeability across Caco-2 cell monolayers. After validation of the use of several drugs simultaneously as internal standards, the usefulness of the method was further probed by testing the possible interactions during absorption between drugs and plant extracts that are used as food supplements, functional foods, or natural laxatives. These extracts contain several active compounds, such as flavonoids, alkyl gallates, or anthraquinones, which are able to partition into the cell membranes and thus affect e.g. the fluidity of the membranes, or diffuse across the cell monolayers, either by using active transport mechanisms or passively.

Five to ten drugs in one experiment at individual 50 ÁM concentrations did not cause problems in cell viability (MTT test) or monolayer integrity (transepithelial electrical resistance (TEER) measurements, and 14C-mannitol diffusion test). The individual permeability values in the cocktails correlated closely with those obtained from single-drug experiments, except when active transport of drugs was involved. Drugs with passive permeability can be included in cocktails; no interactions between them are expected. Drugs, which occupy same binding sites of a transport protein, cannot be included as internal standards. According to the results, standard testing of Caco-2 functionality does not require the use of more than 3-4 compounds with very low, low, moderate and high permeability (including substrates for transporters), depending on the characteristics of the studied drug candidates.
To probe the effects of plant extracts on the permeability of co-administered drugs, standard drugs with known absorption behaviour can be used. Flax seed extract, for example, decreased the permeability of all co-administered drugs. When flax seeds are used as a laxative, fluids with dissolved drugs are adsorbed on the fibers of flax seed, leading to decreased drug absorption. The effects of other plant extracts, which contain high concentrations of different flavonoids and alkyl gallates, are difficult to predict, because their interactions may be mediated via several active transport proteins, such as OCT, MDR1 and different MRP┤s, or additionally via the effects on the cell membrane fluidity, as the permeability experiments with different flavonoids and alkyl gallates proved.

Whereas several food-drug interactions have often been attributed to the inhibition of drug metabolizing enzymes, information regarding the effects of food components on transporters during absorption, distribution and excretion of drugs is still limited. Caco-2 cell monolayers, when expressing active transport and efflux proteins, are therefore very well suitable as in vitro model for this type of studies.